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PubMedDate not supplied

[Nuclear RNP, containing pre-mRNA. XIV. Nature of particles, containing 5'- and 3'-end structures].

Some characteristic peculiarities of the 5'-end and 3'-end structures of pre-mRNA isolated from nuclear RNP particles have been investigated: presence of triphosphorylated nucleotides on the 5'-ends, as a characteristic of the primary product of transcription; presence of modified 5'-ends -- blocked and methylated structures -- caps; presence of poly(A) blocks attached to the 3'-end of pre-mRNA during post-transcriptional transformation of the latter. It was shown that pre-mRNA isolated from nuclear RNP particles contained triphosphorylated nucleotides as well as a "cap" structure at the 5'-ends. On the 3'-ends of pre-mRNA from nuclear RNP particles isolated in the presence of a RNAse inhibitor, the presence of poly(A) blocks have been shown. These poly(A) structures are separated very easily from pre-mRNA during mild RNAase digestion. This is the reason why they are not detected in 30S monoparticles, containing pre-mRNA fragments connected with one informofer. Almost all poly(A) complexes in this condition are combined with proteins and have the sedimentation coefficient of about 14S. It was concluded that formation of nuclear pre-mRNA containing RNP-particles take place just after the onset of RNA synthesis. All processing steps occur with pre-mRNA packed in nuclear RNP particles.

Animals↗
PubMed1983

Hybridization of poly(A)-containing ribonucleic acid transported in vitro to cyclic deoxyribonucleic acid-cellulose transcribed from cytoplasmic poly(A)-containing ribonucleic acid.

Isolated nuclei have been employed to study nucleocytoplasmic RNA transport in vitro; however, the specificity of the in vitro process remains largely undefined. To examine this specificity we have employed a hybridization technique that allows recovery of undegraded RNA after hybridization under stringent conditions. Cytoplasmic poly(A)-containing RNA was isolated from rat liver and used as a template for synthesis of cDNA-cellulose. Rat liver nuclei were isolated and incubated in vitro, and the released poly(A)-containing RNA was hybridized to the cDNA-cellulose transcribed from authentic cytoplasmic RNA. A significant portion hybridized (18.5 +/- 4.4%), comparable to the portion of homologous cytoplasmic poly(A)-containing RNA that hybridized (20.4 +/- 2.3%), indicating a considerable sequence homology between these populations. Similar results were obtained when cDNA was transcribed from RNA which was transported in vitro in 45S ribonucleoprotein (21.6%). Control nuclear RNA hybridized to a significantly smaller extent (9.1 +/- 2.7%), and the percentage hybridizing decreased markedly under conditions that allowed nuclear RNA processing but not transport (to 4.3 +/- 2.1%), showing that the hybridization was not due to nonspecific leakage of nuclear RNA. This technique should prove valuable for following in vitro processing of specific probes and for enrichment of sequences differentially transported in vitro following acute injury or carcinogen treatment of rats.

Animals↗
PubMed1983

Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetylcholinesterase.

Neurons in the rat central nervous system (CNS) were examined for their content of both the acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT; acetyl-CoA, choline O-acetyl-transferase, EC 2.3.16), and the transmitter-degrading enzyme, acetylcholinesterase (AChE). ChAT was localized immunohistochemically and AChE was localized histochemically in normal, colchicine-treated, or diisopropylfluorophosphate (DFP)-treated rats, either in neighboring sections by standard procedures or simultaneously in the same sections by immunofluorescence for ChAT, followed by photography, followed by histochemistry for AChE, followed by brightfield rephotography of the same neurons. This combination of fluorescence and brightfield procedures allows both to be carried out at maximum intensity without fear of interference and enables the unambiguous visualization of all neurons that might be expected with either procedure. Based on previous examination of singly stained neighboring sections, five CNS areas were examined in detail in DFP-treated rats using the simultaneous procedure for localizing both ChAT and AChE in the same neurons in the same sections. Several hundred neurons were thus examined in each area. These areas were the caudate putamen, nucleus basalis of Meynert, medial septum, nucleus of the diagonal band of Broca (vertical and horizontal limbs), and zona incerta. In the caudate putamen and nucleus basalis magnocellularis of Meynert, all ChAT-positive neurons contained AChE and vice versa. In the medial septum and nucleus of the diagonal band of Broca, all ChAT-positive neurons contained AChE, but these neurons containing both ChAT and AChE were intermingled with neurons positive only for AChE.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMedDate not supplied

A testing and evaluation protocol for in-hospital sterilization containers and its application to a new container system.

A new containerized sterilization system has been designed to fulfill the needs of central supply departments and operating rooms, as well as the recommendations of various professional associations and accrediting agencies. The testing that was performed to demonstrate the function and efficacy of the system in the hospital environment illustrates the evaluation criteria that should be applied to any containerized sterilization system. Among the unique features of this containerized system are: a tortuous-path lid design to protect the permeable biobarrier portion of the container from direct challenge, a drain to allow condensation to exit the container, and reusable valves to allow steam entry and air removal. Evaluation of containers with reusable valves revealed the cause of a previously unexplained phenomenon that affects drying and suggested means by which this phenomenon can be prevented.

Central Supply, Hospital↗
PubMed1997

Synthesis, DNA binding, cytotoxicity and sequence specificity of a series of imidazole-containing analogs of the benzoic acid mustard distamycin derivative tallimustine containing an alkylating group at the C-terminus.

In an attempt to produce additional alkylation and crosslinking in the minor groove of DNA, imidazole-containing analogs of distamycin were synthesized with benzoic acid mustard (BAM) and methoxyaziridinyl moieties present at the N- and C-termini, respectively. Analogs 1a-c differed in the number of methylene units (2-4 respectively) between the C-terminal carbonyl group and the methoxyaziridinyl moiety. DNA binding affinity to several polynucleotides decreased with increasing linker length, whereas DNA interstrand crosslinking ability, as measured by a plasmid gel based assay, increased. The in vitro cytotoxicity in human chronic myeloid leukemia K562 cells and the panel of human tumor cell lines at the National Cancer Institute decreased with increasing number of methylene units, and no increase in cytotoxicity was observed over compound AR-1-122 which did not contain the methoxyaziridinyl moiety. 1a-c had the same sequence selectivity of alkylation as AR-1-122, showing alkylation only at 5'-TTTTGPu sequences. The relative binding to these sequences decreased with increasing number of methylene units. The addition of a methoxyaziridinyl moiety in this group of imidazole and BAM-containing compounds can, therefore, increase crosslinking ability to naked DNA but this does not result in an increase in cytotoxicity. In contrast the cytotoxicity was related to their ability to produce sequence specific alkylation at 5'-TTTTGPu sequences.

Alkylating Agents↗
PubMed1998

cDNA cloning of two splice variants of a human copper-containing monoamine oxidase pseudogene containing a dimeric Alu repeat sequence.

Two alternatively spliced transcripts, psiHLAO1 and psiHLAO2, of a copper-containing monoamine oxidase pseudogene have been isolated from a human-liver cDNA library. The larger psiHLAO1 cDNA (2073bp) contains a 5'-flanking segment of 134bp, followed by an apparent open reading frame (ORF) of 1725bp. The deduced amino acid sequence of this ORF (574 residues) shares 81.0% similarity with the 763-residue monoamine oxidase from human placenta (HPAO) (the N-terminal 533 residues of psiHLAO1 share 86.7% similarity with HPAO). The psiHLAO1 ORF is interrupted by an in-frame stop codon corresponding to amino acid 225 and terminates within a type S(a) dimeric Alu repeat sequence. psiHLAO2 appears to be an alternatively spliced variant of psiHLAO1 that has 413 bases of psiHLAO1 excised according to the 'GT-AG' rule. The slightly longer 3' end of the psiHLAO2 transcript shows that the Alu repeat is followed by an 11-bp poly(A) tract that, in turn, is followed by an AT-rich (81%) sequence of 105bp. A reverse transcriptase-polymerase chain reaction (RT-PCR) protocol was used to confirm that both psiHLAO1 and psiHLAO2 are transcribed in human liver and placenta. A search of the expressed sequence tag (EST) database indicates that, like HPAO, psiHLAO derives also from the region 17q21 of the human genome.

Alternative Splicing↗
PubMed2001

CD150 association with either the SH2-containing inositol phosphatase or the SH2-containing protein tyrosine phosphatase is regulated by the adaptor protein SH2D1A.

CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negative signals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phosphatase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan's disease SH2-protein (DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome and non-Hodgkin's lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines, where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines from X-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated by CD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosine at Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 are essential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1A may function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptor tyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2 subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin, carcinoembryonic Ag, and leukocyte-inhibitory receptor families.

Amino Acid Sequence↗
PubMed1996

Comparison between a cisplatin-containing regimen and a carboplatin-containing regimen for recurrent or metastatic bladder cancer patients. A randomized phase II study.

BACKGROUND: The aim of this randomized Phase II study was to compare the efficacy and toxicity of a cisplatin-containing regimen with a carboplatin-containing regimen for patients with recurrent or metastatic bladder cancer. METHODS: Fifty-seven patients with recurrent or metastatic bladder cancer were randomized to receive M-VEC treatment (methotrexate, vinblastine, epirubicin, and cisplatin) (n = 29) or M-VECa treatment (methotrexate, vinblastine, epirubicin, and carboplatin) (n = 28). The chemotherapy was scheduled at 28-day intervals. Recombinant granulocyte-colony stimulating factors were administered daily when the absolute neutrophil count fell below 1000/mm3. The development of ototoxicity was evaluated by measuring auditory brain stem response. RESULTS: Of the 57 entered patients, 55 were evaluable for response and toxicity. The overall clinical response rate was 71% (with 25% complete responses) in the M-VEC group and 41% (with 11% complete responses) in the M-VECa group (P = 0.04). M-VEC chemotherapy was associated with more pronounced side effects. There was a statistically significant difference between M-VEC and M-VECa in terms of gastrointestinal toxicity (P = 0.04), nephrotoxicity (P = 0.03), and neurotoxicity (P = 0.02) during Cycle 3 of chemotherapy. Leukopenia and neutropenia were worse in the M-VECa arm, but not significantly so (P = 0.4). Ototoxicity was only detected in one of seven examined M-VEC patients after two cycles of chemotherapy. CONCLUSIONS: M-VECa has a low level of gastrointestinal, renal, neurologic, and otologic toxicity, but is apparently less effective than M-VEC in the treatment of recurrent or metastatic bladder cancer. However, a larger, randomized Phase III trial is needed to confirm these results.

Aged↗
PubMedDate not supplied

Investigation of corrosion and container integrity in metal food containers using scanning electron microscopy-X-ray microanalysis.

Scanning electron microscopy and x-ray microanalysis were used to investigate microleakage, two incidences of loss of coating integrity, filiform corrosion, and sulfide black corrosion in enamel-lined metal food containers. Microleakage developed in a canned cheese sauce at the side seam weld and was traced to a hot weld and a cold weld defect. An enamel-blistering problem developed in an aseptically processed milk-based product when the tinplate was heated above its melting point; the tin melted under the enamel coating, causing the blistering. An enamel-blistering problem, due to silicon contamination of the metal surface prior to enameling, developed on the bottom of a two-piece can that contained a chicken-based product. A sulfide black problem developed in canned clams, caused by scratches in the coating that were introduced during can manufacturing. A filiform corrosion problem developed on the outside of the easy score line of enameled tinplate lids with a light tin coating 0.08 microns thick.

Animals↗
PubMed1997

The treatment of Legg-Calvé-Perthes disease. To contain or not to contain.

In order to evaluate a treatment protocol involving a non-containment method and femoral osteotomy when lateral subluxation of the femoral head develops, 39 children with 42 affected hips were examined radiologically an average of 7 years (4-16 10/12) following the onset of Legg-Calvé-Perthes disease. Twenty-six hips were treated conservatively with an ischial-bearing, Thomas-type splint and 16 with a femoral varus derotating osteotomy. Both Catterall and Herring gradings were used. The end result was evaluated according to the Stulberg classification. All Catterall grade I (n = 8) hips had a satisfactory result regardless of age at onset and method of treatment. In Catterall groups II-IV (n = 34), age at onset influenced the treatment outcome. In children affected before the age of 8 years, results of conservative treatment were originally satisfactory but were seen to deteriorate with advancing age. In children over 8 years old (n = 13), the results clearly favour operative containment.

Age Factors↗
PubMed1984

Isoelectrophoretic separation and the detection of soluble proteins containing acid-labile phosphate: use of the phosphoenolpyruvate:sugar phosphotransferase system as a model system for N1-P-histidine- and N3-P-histidine-containing proteins.

Procedures have been developed for the detection of acid-labile phosphorylations of proteins. The phosphoproteins were separated by native isoelectric focusing while maintaining the gel at about 0 degree C, and denaturing urea-Nonidet isoelectric focusing gels were adapted to run at -10 degrees C. The proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), HPr, which contains 1-P-histidine, and factor IIIglc and enzyme I, which contain 3-P-histidine when they are phosphorylated, were used to develop the conditions. Autoradiography of [32P]-labeled phosphoproteins was carried out on frozen gels which had not been acid fixed in order to avoid hydrolysis of the phosphohistidines . The frozen gels were subsequently fixed and stained, and reautoradiography revealed whether the phosphoproteins were acid stable or labile. In addition to the known proteins of the PTS, at least one other protein whose phosphorylation was dependent on enzyme I and HPr was found in Salmonella typhimurium and Escherichia coli [E.B. Waygood , and R.L. Mattoo (1983) Canad . J. Biochem. Cell Biol. 61, 150-153]. Initial experiments with rat tissues have demonstrated acid-labile phosphorylations in proteins which were either [gamma-32P]ATP or [32P]phosphoenolpyruvate dependent. The interconversion of phosphoenolpyruvate and ATP in crude extracts of bacterial cells was examined, and appropriate controls were found. Protein phosphorylation dependent upon phosphoenolpyruvate was much greater in S. typhimurium and E. coli than the corresponding ATP-dependent phosphorylation, while the opposite was found for rat tissues.

Adenosine Triphosphate↗
PubMed1990

Evidence that chemical modification of a positively charged residue at position 189 causes the loss of catalytic activity of iron-containing and manganese-containing superoxide dismutases.

The Escherichia coli, Bacillus stearothermophilus, and human manganese-containing superoxide dismutases (MnSODs) and the E. coli iron-containing superoxide dismutase (FeSOD) are extensively inactivated by treatment with phenylglyoxal, an arginine-specific reagent. Arg-189, the only conserved arginine in the primary sequences of these four enzymes, is also conserved in the three additional FeSODs and five of the six additional MnSODs sequenced to date. The only exception is Saccharomyces cerevisiae MnSOD, in which it is conservatively replaced by lysine. Treatment of S. cerevisiae MnSOD with phenylglyoxal under the same conditions used for the other SODs gives very little inactivation. However, treatment with low levels of 2,4,6-trinitrobenzenesulfonate (TNBS) or acetic anhydride, two lysine-selective reagents that cause a maximum of 60-80% inactivation of the other four SODs, gives complete inactivation of the yeast enzyme. Total inactivation of yeast MnSOD with TNBS correlates with the modification of approximately five lysines per subunit, whereas six to seven acetyl groups per subunit are incorporated on complete inactivation with [14C]-acetic anhydride. It appears that the positive charge contributed by residue 189, lysine in yeast MnSOD and arginine in all other SODs, is critical for the catalytic function of MnSODs and FeSODs.

Amino Acid Sequence↗
PubMed1977

Increased synthesis of abundant poly(A)-containing RNA in a DNA defective mutant of Saccharomyces cerevisiae containing the "killer character".

A Saccharomyces cerevisiae strain which contains both the "killer character" and a ts mutation in the initiation of nuclear DNA synthesis (cdc4) was studied. Incubation of this strain at the restrictive temperature caused a 3--4 fold increase in the relative rate of synthesis of abundant RNA which contains poly(A) and a 2--3-fold increase in the relative rate of synthesis of killer dsRNA. Thus, the amount of killer dsRNA found in these cells seems to be correlated to the amount of abundant poly(A)-RNA.

DNA↗
PubMed1989

Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens.

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2', 5'-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens. The latter and the form 2 enzyme share 11 identical residues in the NH2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.

Amino Acid Sequence↗
PubMed1984

A new type of carbohydrate-containing synthetic antigen: synthesis of carbohydrate-containing polyacrylamide copolymers having the specificity of O:3 and O:4 factors of Salmonella.

The synthesis of a new type of synthetic antigen that contains no protein is described. Two linear polyacrylamide copolymers with carbohydrate branches were obtained via radical copolymerisation of the allyl glycosides of the oligosaccharide determinants O-beta-D-mannopyranosyl-(1----4)-O-alpha-L-rhamnopyranosyl-(1----3 )-beta-D- galactopyranose and O-3,6-dideoxy-alpha-D-xylo-hexopyranosyl-(1----3)-alpha-D-mannopyranose with acrylamide. These copolymers, which contained 30% of carbohydrate and had molecular masses exceeding 100 kilodaltons, had the group specificity E and B of Salmonella.

Antigens, Bacterial↗
PubMed1984

The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene.

The family of approximately 16 genes encoding the mouse ribosomal protein L32 has been characterized by an analysis of a representative set of genomic clones. Surprisingly, this family contains only a single expressed intron-containing gene. This gene, termed rpL32 , has been completely sequenced and found to possess certain novel features including the presence in two of its introns of a sequence with high homology to the 5' end of U1 snRNA and a 5' terminal region exceptionally rich in pyrimidines. Most of the other members of the L32 family appear to be processed genes, some of which are identical or very similar to the rpL32 gene, except for the lack of introns. One unmutated gene was found to be integrated 28 nucleotides downstream of a canonical TATA box. However, despite this feature, the gene does not seem to be expressed, as judged by its extent of methylation compared to the expressed rpL32 gene.

Amino Acid Sequence↗
PubMed1992

The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages.

We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.

Animals↗
PubMed1986

Promotion of cell-substratum adhesion of clonal rat pheochromocytoma cells (PC12) by factors contained in glioma-conditioned medium (GCM): separation of two active factors contained in GCM.

Culture medium conditioned over C6 glioma cells (GCM) contains factors which induce neurite outgrowth from clonal rat pheochromocytoma (PC12) cells. The effects of GCM on the cell-substratum adhesion of PC12 cells, which is an early event required for the neurite outgrowth, were investigated. The results obtained are as follows. Addition of GCM promoted the adhesion of PC12 cells specifically to collagen-coated tissue culture dish. The GCM-promoted adhesion of PC12 cells was prevented by the treatment of the cells with cytochalasin B, concanavalin A and glycosidase mixture suggesting the contribution of microfilaments and cell surface carbohydrates in the cell adhesion. GCM did not increase significantly the intracellular content of cAMP and the extent of cell adhesion promoted by cAMP or dibutyryl-cAMP was much less than that by GCM. Two active factors contained in GCM were separated by either gel filtration or chromatofocusing using the cell adhesion assay as an index. The first factor with an apparent mol. wt. around 40,000 had the abilities to induce the neurite outgrowth and to enhance the choline acetyltransferase activity in addition to the ability to promote the adhesion of PC12 cells. The second factor with an apparent mol. wt. around 10,000 was devoid of the ability to induce the neurite outgrowth, but had the abilities to enhance the choline acetyltransferase activity and to promote the adhesion of PC12 cells. Both factors were sensitive to trypsin digestion and relatively heat stable. The significance of these factors in the neuronal differentiation was discussed.

Adrenal Gland Neoplasms↗